This application addresses the compelling need to develop new and improved vaccine adjuvants for stimulating protective immunity at mucosal surfaces. The proposal involves a consortium arrangement between the University of Louisville, the University at Buffalo, and the Hauptman-Woodward Institute, and brings together complementary and integrated expertise in vaccine adjuvant research, structural and molecular biology, and induction of mucosal immunity. Studies by this group have succeeded to dissociate useful adjuvant properties from the toxicity of LT-IIb, an AB5-type heat-labile enterotoxin of Escherichia coli. Specifically, the recombinantly expressed B pentameric subunit of LT-IIb (LT-IIb-B5) not only lacks enterotoxicity but, strikingly, can activate the Toll-like receptor (TLR)-2/TLR1 signaling complex and stimulate antibody responses to co-administered vaccine proteins. Proof-of-concept preliminary studies have shown that certain engineered point-substitution mutations in the active region of LT-IIb-B5 enhance its ability to interact with TLR2 and TLR1. The overall objective of this application is to better characterize the physical and functional interaction of LT-IIb-B5 with the TLR2/TLR1 heterodimer, engineer improved versions of LT-IIb-B5 in terms of TLR2/TLR1-mediated adjuvanticity, and develop potential adjuvants through studies in preclinical models of vaccination against oral infection. This will be accomplished through appropriate and innovative molecular biological, structural, and immunological studies outlined in the following four specific aims: 1) To rationally engineer enhanced TLR2-interactive versions of LT-IIb-B5. 2) To structurally characterize the LT-IIb- B5-TLR2/TLR1 interaction using small-angle X-ray scattering (SAXS). 3) To evaluate the engineered LT-IIb-B5 mutants for in vitro TLR2/TLR1-dependent immunostimulatory activities. 4) To determine the ability of LT-IIb-B5 and improved engineered versions thereof to induce protective immunity in vivo. The generated data will form the basis for the long-term goal which is to establish LT-IIb-B5 and improved engineered derivatives as effective adjuvants in vaccine formulations against pathogens which colonize or invade via oral, respiratory, or urogenital mucosal surfaces.